The present invention relates to isolated nucleic acid sequences encoding polypeptides having proteolytic activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
Detergent proteases have been developed by isolation of proteases found in nature go followed by testing in detergent formulations. Most detergent proteases are obtained from members of the genus Bacillus.
Examples of commercial protease products are Alcalase(copyright), Esperase(copyright) and Savinase(copyright), all supplied by Novo Nordisk A/S, Denmark. These and similar enzyme products from other commercial sources are active in detergent solutions, i.e. at pH values in the range of from 8 to 11 and in the presence of sequestering agents, surfactants and bleaching agents such as sodium borate. The Alcalase(copyright) protease is produced by strains of the species Bacillus licheniformis. The Esperase(copyright) and Savinase(copyright) proteases are obtained by cultivation of strains of alkalophilic Bacilli.
WO 92/17577 describes a protease isolated from Bacillus sp. TY145, NCIMB 40339. The isolated protease had a pH optimum in the range of from 8 to 11, a temperature optimum in 30 the range of from 45 to 55 C., a pl around 8.8, and an apparent molecular weight of about 38 kD. The gene producing the above-mentioned protease has now been cloned and expressed in Bacillus subtilis. Thus, it is an object of the present invention to provide isolated nucleic acid sequences encoding polypeptides having proteolytic activity a well as variants of said protease.
In a first aspect, the present invention relates to an isolated nucleic acid sequence encoding a polypeptide having proteolytic activity, selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 75% identity with amino acids 1 to 311 of SEQ ID NO: 2;
(b) a nucleic acid sequence having at least 70% identity with nucleotides 371 to 1303 of SEQ ID NO: 1;
(c) a nucleic acid sequence, which hybridizes under low stringency conditions with
(i) the nucleic acid sequence of SEQ ID NO: 1,
(ii) a subsequence of (i) of at least 100 nucleotides, or
(iii) a complementary strand of (i) or (ii);
(d) an allelic variant of (a), (b), or (c); and
(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has proteolytic activity.
In a second aspect, the present invention relates to a variant of the polypeptide having the amino acid sequence shown as amino acids 1 to 311 of SEQ ID NO: 1, which comprises at least one modification compared to amino acids 1 to 311 of SEQ ID NO: 1 and which has at least 75% identity with amino acids 1 to 311 of SEQ ID NO: 1.
In other aspects, the present invention also relates to detergent compositions comprising such variants, nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.